The invention relates to improved polynucleotide probe configurations for detecting structural abnormalities that result from chromosome breakage and rearrangement, particularly as used in the detection of several types of genetic disorders related to cancer and other diseases. The invention further relates to an improved method of detecting translocations using probe sets which span each breakpoint region associated with a translocation and the regions on both sides beyond the 3xe2x80x2 and 5xe2x80x2 ends of each breakpoint region.
A number of inherited genetic diseases and types of cancer have been linked to chromosomal translocation events which result in the fusion of two genes which do not occur together in the normal genome. Certain conditions involve translocations which frequently occur at the same or very near location. The chromosome regions where frequent breaks occur are called breakpoint regions.
One of the best known examples of a clinically important translocation is the Philadelphia Chromosome which results from a break in the ABL1 gene on distal chromosome 9q and the BCR gene on proximal chromosome 22q {t(9;22)} (FIG. 1). The breakpoints within the ABL1 gene may occur throughout a region spanning more than 175 kb upstream from exon II while the breaks in chromosome 22 are clustered into two areas of the BCR gene, termed the major breakpoint cluster region (m-bcr) and the minor breakpoint cluster region (M-bcr) (Kurzrock et al, New England Journal of Medicine, 319:990 (1988)). The Philadelphia Chromosome occurs in most cases of Chronic Myelogenous Leukemia (CML) and some cases of Acute Lymphocytic Leukemia (ALL). Other important translocations include, but are not limited to, t(8;21) in Acute Myelogenous Leukemia, t(8:14) in Burkett""s Lymphoma and pre-B-cell Acute Lymphoblastic Leukemia, t(1:14), t(7:9), t(7:19), t(11:14), t(10:14) and t(7:9) in T-acute Lymphoblastic Leukemia, t(15;17) in Acute Myelogenous Leukemia (AML) and t(15:17) Acute Promyelocytic Leukemia (PML). Solid tumors include, t(9;22) in Ewing""s Sarcoma, t(15:16), and hereditary diseases associated with translocations include a number of mental retardation associated syndromes. It is likely that other conditions are caused by subcriptic translocations or other structural aberrations which are yet to be determined and are too small to be noticed by standard cytogenetics.
Multiple genetic testing methods have been developed for use in diagnosis, monitoring of minimal residual disease and/or response to therapy during clinical practice. However, no single technique has been developed that can accurately detect and quantify disease at diagnosis and throughout treatment. Conventional quantitative cytogenetics and G-banding analysis is cumbersome and can only be applied to cycling cells (Lion, Leukemia 10: 896 (1996)). In practice, the sensitivity of conventional cytogenetics is dependent upon the number of good metaphase cells which can be evaluated. In the example of cancers caused by neoplastic cells in the bone marrow, obtaining large numbers of good metaphase cells from bone marrows of patients is difficult.
More recently, the assay technique in situ hybridization (ISH), particularly fluorescent in situ hybridization (FISH) (Pinkel et al, Proc. Natl. Acad. Sci., U.S.A. 83:2934-2938 (1986)) has been of assistance in detecting translocations. FISH allows the analysis of individual metaphase or interphase cells, thereby eliminating the need to obtain and assay cycling cells. It is therefore possible to use nondividing tissue, including bone marrow and peripheral blood cells in a diagnostic or prognostic analysis.
In the field of detecting the Philadelphia Chromosome, a commonly used method for detection of ABL1/BCR fusion utilizes differently labeled probes for BCR and ABL1, and detects a single ABL1/BCR fusion (or closely linked) signal in cells with a Ph chromosome. (This method is referred to for convenience as S-FISH.) An example of this technique is Tkachuk et al, Science 250: p. 559-562 (1990) where one fluorescently labeled probe hybridized to part of the ABL1 gene and a second fluorescently labeled probe hybridized to part of the BCR gene.
The probes in commercial single FISH test kits do not span the entire length of each translocation breakpoint but rather are designed to bind to one portion of each gene, i.e. sometimes overlapping or adjacent to a breakpoint region, sometimes many kilobases away and sometimes both (See FIG. 1 of Tkachuk et al for example). Normal chromosomes 9 and 22 each bind one probe, which is specific to that chromosome. The Philadelphia Chromosome, both probes hybridize at the fusion site bringing both labels in close proximity so as to usually form a color shift or fusion near proximity/signal. Because the exact breakpoint may vary, the two probe labels may not come sufficiently close to form a fusion label. Likewise for probes useable to detect the t(8;21) translocation in Acute Myelogenous Leukemia (AML).
Using the probe configuration above, the following detection method for the Philadelphia Chromosome using FISH has been used: the ABL1 gene probe is labeled using a probe containing one hapten or fluorophore (for example, FITC) and the BCR gene probe is labeled using a probe containing another hapten or fluorophore (for example Rhodamine). After hybridization and detection, a normal chromosome 9 shows the green signal and a normal chromosome 22 shows a red signal. A normal cell would therefore exhibit two red signals and two green signals. A cell containing a Philadelphia chromosome has one red and one green signal for the unaffected homologues of chromosomes 9 and 22 and one white, yellow or closely linked pair of signals that results from the close proximity of the labeled probes hybridized to the translocated BCR and ABL1 genes, the so-called fusion signal.
However, the probes used heretofore in this method have not been constructed so as to specifically bind and detect the second fusion site for the reciprocal translocation event. Thus, the S-FISH method detects only one of the abnormal chromosomes resulting from the translocation event, the Philadelphia chromosome.
In another method using labelled probes to detect ALL gene rearrangements in solid tumors, a probe set was designed so that the two probes lie adjacent to each other on the normal chromosome, but split apart and move to the two different abnormal chromosomes if the translocation has occurred (Croce, U.S. Pat. No. 5,567,586, hereby incorporated by reference). In this method the probes are designed to be complementary to sequences in the translocation region on one chromosome. In this method, the fluorescent probes produce a single spot on the normal chromosome, but appear as two distinct spots when translocation has occurred.
The same format has been used for other assays for detecting other translocations such as t(8:21) in Acute Myeloid Leukemia (AML). For example, Le Beau, Blood 81: 1979-1983 (1993), and Sacchi et al, Cancer Genetics and Cytogenetics 79: 97-103 (1995) and Fischer et al, Blood 88: 3962-3971 (1996).
It is an object of the invention to provide methods with increased sensitivity and accuracy for detecting chromosome translocations and other structural rearrangements which result in more than one abnormal fusion site in the genome.
It is a further object of the invention to provide probes and probe sets which are useful in detecting reciprocal genetic translocations according to the methods of the invention.
It is another object of the present invention to detect cancer, inherited disease, susceptibility to inherited disease or a carrier of a fused gene for an inherited disease wherein the condition results from a chromosomal translocation in one or more cells. This is particularly beneficial when the diagnosis, prognosis, monitoring for residual disease and response to therapy in cancer or other disease is dependant upon the quantity of abnormal cells as an indicia of the disease state and/or response to treatment.
It is also an object of the invention to provide a means of constructing such probes and probe sets, which will detect reciprocal fusions resulting from chromosomal translocations and will accordingly be useful in diagnosis, prognosis, monitoring of residual disease and response to therapy when reciprocal chromosome translocations are present.
It is still another object of the present invention to provide diagnostic test kits which can be used by any cytogenetist or other trained individual to detect multiple fusion events which result from structural rearrangement of the genome.
Probes and probe sets of the present invention have the characteristic of encompassing the entire breakpoint region and a region on each side of the breakpoint region on each chromosome for the reciprocal translocation event of interest and are capable of detecting such translocations with much greater sensitivity than the probes and probe sets which were previously known.
A particularly preferred probe set and method is used for detecting the Philadelphia chromosome and its corresponding derivative chromosome as companion indicators of CML and some other cancers such as ALL. One functional probe is designated P5161-DC, described hereinbelow. Another example is for detecting the AML1/ETO gene fusion in AML.
The use of specifically designed probe sets by the method of the present invention has allowed the clinician to assess physical information regarding all fusion events associated with a defined structural rearrangement in a cell. For example, using the standard detection method of fluorescence in situ hybridization (FISH) it has been demonstrated that these probe sets provide the following advantages over traditional testing methodologies for detecting the same translocation.
1. Unlike traditional single fusion probe sets, probe sets which detect multiple, derivatives of a structural rearrangement have the ability to detect much lower copy numbers of abnormal cells thereby providing greater improved diagnostics using FISH assays
2. The ability of the probe sets to derive necessary information from cells in interphase, Thereby rivaling the sensitivity of metaphase cells in conventional cytogenetics.
3. Specifically, increased sensitivity has been demonstrated with multiple fusion probes used in interphase FISH analysis which is at least as sensitive as Q-cytogenetics (the previous gold standard) for monitoring bone marrow or peripheral blood cell populations for minimal residual disease and response to therapy.
4. Greater sensitivity allows the use of peripheral blood instead of invasive and painful bone marrow samples from patients for routine testing, to monitor for minimal residual disease and response to therapy.
5. By detecting high and low copy numbers of gene fusions, the present invention can be used for diagnosis and monitoring throughout the course of the disease thereby avoiding traditional multiple assay-type testing methodologies.
6. Simplified sample requirements and testing provides further benefits in cost and patient well being.